Anti cancer combination therapy

ABSTRACT

The invention relates to the combined use of certain bispecific, VEGF and Ang2 binding molecules with PD1 antagonists for the treatment of cancer. It further relates to pharmaceutical compositions and kits comprising such binding molecules and antagonists.

FIELD OF THE INVENTION

The present invention relates to a combination therapy in the treatmentof cancer and to compounds for use in such a combination therapy. Thecompounds for combination are a bispecific binding molecule, like anantibody derivative, and a PD-1 antagonist.

BACKGROUND OF THE INVENTION

Angiogenesis is the development of new blood vessels to provide anutritive blood supply and a prerequisite for solid tumor growth andsurvival. Approaches in cancer therapy have been made in which keymolecules involved in angiogenic pathways are targeted. These includemodes of inhibition of the vascular endothelial growth factor (VEGF)signaling pathway as VEGF is considered the most potent angiogenicgrowth factor.

Lung cancer is the leading cause of cancer-related mortality with nearly1.6 million deaths worldwide in 2012 or nearly 20% of cancer mortalityas a whole (Molina et al., Mayo Clin Proc. 2008; 83(5):584-594; Chan etal., Transl Lung Cancer Res. 2014; 4(1):36-54). The most common type oflung cancer is the non-small cell lung cancer (NSCLC), which accountsfor approximately 85% of all lung cancers (Chen et al. Nat Rev Cancer2014; 14:535-546; Nawaz et al. Nat Rev Drug Discov. 2016; 15:229-230).NSCLC is nowadays understood as comprising a heterogeneous set ofdiseases having diverse pathophysiological characteristics, among thempulmonary adenocarcinoma, squamous-cell carcinoma (SCC) and large cellcarcinoma as the most prominent subtypes.

In recent years, diverse new treatment options for patients with locallyadvanced or metastatic disease at the time of diagnosis in addition tochemotherapy and radiation therapy have been developed for cancer,especially for NSCLC. These new treatment methods are targeted therapiesinvolving e g small molecule inhibitors or receptor monoclonalantibodies (mAb) and are based on alterations of the majorcell-signaling and regulatory pathways—including alterations in receptortyrosine kinases (TKs), such as epidermal growth factor receptor (EGFR),and alterations in angiogenic pathways—that are frequent in lung cancer.Much of recent work has focused on mutations of EGFR and on abnormalfusion of anaplastic lymphoma kinase (ALK) being inhibited by EGFRtyrosine kinase inhibitors and crizotinib. It has further been shownthat combining the administration of the monoclonal antibodybevacizumab, which targets VEGF, with chemotherapy resulted in asignificant improvement in survival among patients with colorectalcancer (Hurwitz et al. New Eng J Med 2004; 350:2335-2342) or advancednon-squamous NSCLC (Sandler et al. J Clin Oncol 2005 23 (16 s pt 1):2s). In addition to therapies of blocking components directly involved inthe VEGF pathway, e.g. Angiopoietin2 (Ang2), a ligand of the Tie2receptor tyrosine kinase controlling vascular remodeling by enabling thefunctions of other angiogenic factors, such as VEGF, has been anotherappealing target in cancer therapy.

WO2010/040508 and Kienast et al. (Clin Cancer Res; 19(24), 2013)disclose vanucizumab, a bispecific anti-VEGF/anti-Ang-2 antibody and itsuse in the treatment of cancer.

EP2694546 B1 and WO2012/131078 A1 relate to a bispecific bindingmolecule comprising a VEGF-binding immunoglobulin single variable domainand an Ang2-binding immunoglobulin single variable domain, and further aserum albumin binding immunoglobulin single variable domain that is usedin the treatment of cancer and other diseases.

WO2016/170039 A1 relates to a combination therapy of an antibodyspecifically binding to Angiopoietin 2 with an antibody specificallybinding to programmed death 1 polypeptide (PD-1).

WO2016/170040 A1 relates to a combination therapy of an antibodyspecifically binding to Angiopoietin 2 and an antibody specificallybinding to VEGF with an antibody specifically binding to programmeddeath ligand 1 (PD-L1).

Despite all the approaches, there is still a need for improved treatmentoptions for cancer patients. It is therefore an object of the presentinvention to provide pharmaceutical compositions and methods in cancertherapy for improved therapeutic efficacy and applicability.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for treating a patient with abispecific anti-VEGF/anti-Ang-2 antibody together with an antagonistagainst Programmed Death 1 (PD-1)—an immunoinhibitory protein thatnegatively regulates T cell receptor signals. The treatment leads to asignificant reduction of tumor growth or even to tumor shrinkage (FIG.1). Accordingly, the present invention provides a combination therapycomprising a bispecific anti-VEGF/anti-Ang-2 antibody and a PD-1antagonist.

In a detailed aspect, the present invention relates to a method oftreating and/or preventing an oncological or hyperproliferative disease,in particular cancer or a tumor disease, comprising administering to apatient in need thereof

-   a) a therapeutically effective amount of Compound A, and-   b) a therapeutically effective amount of Compound B,    -   wherein        -   Compound A is a bispecific binding molecule comprising            -   a VEGF-binding immunoglobulin single variable domain,            -   a serum albumin binding immunoglobulin single variable                domain, and            -   an Ang2-binding immunoglobulin single variable domain        -   wherein            -   said VEGF binding immunoglobulin single variable domain                has the following CDR sequences:

CDR1: (SEQ ID NO: 1) SYSMG CDR2: (SEQ ID NO: 2) AISKGGYKYDAVSLEG CDR3:(SEQ ID NO: 3) SRAYGSSRLRLADTYEY

-   -   said serum albumin binding immunoglobulin single variable domain        has the following CDR sequences:

CDR1: (SEQ ID NO: 4) SFGMS CDR2: (SEQ ID NO: 5) SISGSGSDTLYADSVKG CDR3:(SEQ ID NO: 6) GGSLSR,

-   -   said Ang2-binding immunoglobulin single variable domain has the        following CDR sequences:

CDR1: (SEQ ID NO: 7) DYAIG CDR2: (SEQ ID NO: 8) AIRSSGGSTYYADSVKG CDR3:(SEQ ID NO: 9) VPAGRLRYGEQWYPIYEYDAand

-   -   wherein        -   Compound B is a PD-1 antagonist.

In a related aspect, the present invention provides a Compound A andCompound B, each for use in a method of treating and/or preventing anoncological or hyperproliferative disease, said method comprisingadministering Compound A and Compound B to a patient in need thereof.

The present invention further relates to the use of Compound A andCompound B, each for preparing a pharmaceutical composition for treatingand/or preventing an oncological or hyperproliferative disease, whereinCompound A and Compound B, are intended for or provided for combinedadministration of Compound A and Compound B.

In another aspect, the present invention discloses a pharmaceuticalcomposition comprising

-   -   a) Compound A and    -   b) Compound B    -   wherein        -   Compound A is a bispecific binding molecule comprising            -   a VEGF-binding immunoglobulin single variable domain,            -   a serum albumin binding immunoglobulin single variable                domain, and            -   an Ang2-binding immunoglobulin single variable domain,        -   wherein            -   said VEGF binding immunoglobulin single variable domain                has the following CDR sequences:

CDR1: (SEQ ID NO: 1) SYSMG CDR2: (SEQ ID NO: 2) AISKGGYKYDAVSLEG CDR3:(SEQ ID NO: 3) SRAYGSSRLRLADTYEY

-   -   said serum albumin binding immunoglobulin single variable domain        has the following CDR sequences:

CDR1: (SEQ ID NO: 4) SFGMS CDR2: (SEQ ID NO: 5) SISGSGSDTLYADSVKG CDR3:(SEQ ID NO: 6) GGSLSR,

-   -   said Ang2-binding immunoglobulin single variable domain has the        following CDR sequences:

CDR1: (SEQ ID NO: 7) DYAIG CDR2: (SEQ ID NO: 8) AIRSSGGSTYYADSVKG CDR3:(SEQ ID NO: 9) VPAGRLRYGEQWYPIYEYDAand

-   -   wherein        -   Compound B is a PD-1 antagonist.

In a further aspect, the present invention relates to a kit comprising

-   -   a) a first pharmaceutical composition comprising Compound A and    -   b) a second pharmaceutical composition comprising Compound B,        wherein Compound A and Compound B are defined as above.

DESCRIPTION OF THE FIGURES

FIG. 1 shows tumor growth inhibition upon treating 9 test individuals(open triangles, lower lines) with the bispecific binding moleculeVEGFANGBII22 (compound A) (dose: 15 mg/kg; schedule: every 3-4 days)(A-B) and the CrossMab anti-VEGF/anti-Ang-2 antibody (vanucizumab)(dose: 15 mg/kg; schedule: every 3-4 days) as defined in WO2010/040508or Kienast et al. (2013, supra) (C-D), respectively, in combination withthe rat IgG2a anti-murine PD-1 antibody EX 101359 (clone RMP1-14) (dose:10 mg/kg; schedule: every 3-4 days), as compared to 9 untreatedindividuals (filled circles, upper lines). Treated individuals werefurther administered with the small molecule tyrosine kinase inhibitorvatalanib (EXBF003) (dose: 100 mg/kg; schedule; once per day), to mimicanti-VEGF activity in mice models. Treatment started at day 5 afterinjection of 5×10⁴ LL/2 subcutaneous tumor cells per mouse individual.Indicated are the tumor volume in mm³ (A, C) and the correspondingrandomized, relative tumor volume (B, D), respectively, until day 35 ofthe experiment.

FIG. 2 shows mean survival of mice treated with control/Isotypeantibody, anti-PD1 antibody (dose: 10 mg/kg; schedule: every 3-4 days),VEGFANGBII22 (dose: 15 mg/kg; schedule: every 3-4 days), vatalanib(dose: 100 mg/kg; schedule; once per day), vatalanib plus anti-PD1antibody, vatalanib plus anti-PD1 antibody plus VEGFANGBII22, oranti-PD1 antibody plus VEGFANGBII22. Each treatment group consisted of10 individual tumor bearing randomized mice. Treatment started at day 3after injection of 5×10⁴ LL/2 subcutaneous tumor cells per mouseindividual.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method, compounds for use, use ofcompounds, pharmaceutical compositions and kits, all referring to thecombined therapy or combined provision of Compound A and Compound B,wherein

-   -   Compound A is a bispecific binding molecule comprising        -   a VEGF-binding immunoglobulin single variable domain,        -   a serum albumin binding immunoglobulin single variable            domain, and        -   an Ang2-binding immunoglobulin single variable domain    -   wherein        -   said VEGF binding immunoglobulin single variable domain has            the following CDR sequences:

CDR1: (SEQ ID NO: 1) SYSMG CDR2: (SEQ ID NO: 2) AISKGGYKYDAVSLEG CDR3:(SEQ ID NO: 3) SRAYGSSRLRLADTYEY

-   -   said serum albumin binding immunoglobulin single variable domain        has the following CDR sequences:

CDR1: (SEQ ID NO: 4) SFGMS CDR2: (SEQ ID NO: 5) SISGSGSDTLYADSVKG CDR3:(SEQ ID NO: 6) GGSLSR,

-   -   said Ang2-binding immunoglobulin single variable domain has the        following CDR sequences:

CDR1: (SEQ ID NO: 7) DYAIG CDR2: (SEQ ID NO: 8) AIRSSGGSTYYADSVKG CDR3:(SEQ ID NO: 9) VPAGRLRYGEQWYPIYEYDAandwherein

-   -   Compound B is a PD-1 antagonist.

The present inventors have surprisingly found that a combination ofCompound A and Compound B results in a significant reduction, or evenshrinkage, of tumor growth as compared to a therapy with Compound A onlyor Compound B only. Compound A and B work together synergistically andcan lead to a reduction of cancer.

Compound A according to the present invention is a bispecificanti-VEGF/anti-Ang-2 binding molecule. Anti-angiogenesis therapies havebecome an important treatment option for several types of tumors. Thesetherapies have focused on blocking the VEGF pathway (Ferrara et al., NatRev Drug Discov. 2004; 3(5):391-400) by neutralizing VEGF or itsreceptors. Recent studies in mice have shown that Angiopoietin2 (Ang2),a ligand of the Tie2 receptor, controls vascular remodeling by enablingthe functions of other angiogenic factors, such as VEGF. Ang2 isprimarily expressed by endothelial cells, strongly induced by hypoxiaand other angiogenic factors and has been demonstrated to regulate tumorvessel plasticity, allowing vessels to respond to VEGF and FGF2(Augustin et al., Nat Rev Mol Cell Biol. 2009; 10(3):165-77). Consistentwith this role, the deletion or inhibition of Ang2 results in reducedangiogenesis (Gale et al., Dev Cell. 2002; 3(3):302-4) (Falcon et al.,Am J Pathol. 2009; 175(5):2159-70). Elevated Ang2 serum concentrationshave been reported for patients with colorectal cancer, NSCLC andmelanoma (Goede et al., Br J Cancer. Oct. 26, 2010; 103(9):1407-14),(Park et al., Chest. 2007; 132(1): 200-6), (Helfrich et al., Clin CancerRes. 2009 15; 15(4):1384-92). In CRC cancer Ang2 serum levels correlatewith therapeutic response to anti-VEGF therapy.

Ang2 is a secreted, 66 kDa ligand for the Tie2 receptor tyrosine kinase(Augustin et al., Nat Rev Mol Cell Biol. 2009; 10(3):165-77). Ang2consists of an N-terminal coiled-coil domain and a C-terminalfibrinogen-like domain, the latter is required for Tie2 interaction.Ang2 is primarily expressed by endothelial cells and strongly induced byhypoxia and other angiogenic factors, including VEGF. Tie2 is found onendothelial cells, haematopoietic stem cells and tumor cells. Ang2-Tie2has been demonstrated to regulate tumor vessel plasticity, allowingvessels to respond to VEGF and FGF2.

The Ang-Tie system consists of 2 receptors (Tie1 and Tie2) and 3 ligands(Ang1, Ang2 and Ang4) (Augustin et al., Nat Rev Mol Cell Biol. 2009;10(3):165-77). Tie2, Ang1 and Ang2 are the best studied members of thisfamily, Tie1 is an orphan receptor and the role of Ang4 for vascularremodeling still needs to be defined. Ang2 and Ang1 mediate opposingfunctions upon Tie2 binding and activation. Ang2-mediated Tie2activation results in endothelial cell activation, pericytedissociation, vessel leakage and induction of vessel sprouting. Incontrast to Ang2, Ang1 signaling maintains vessel integrity byrecruitment of pericytes, thereby maintaining endothelial cellquiescence.

Bispecific anti-VEGF/anti-Ang-2 binding molecules that can be usedaccording to the invention are e.g. disclosed in WO2012/131078,incorporated herein by reference).

Compound B according to the present invention is an antagonist against amember of the protein Programmed Death 1 (PD-1) family, such as PD-1itself or one of its ligands, PD-L1 or PD-L2. PD-1 is known as animmunoinhibitory protein that negatively regulates TCR signals (Ishida,Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (2006) Immunol.Immunother. 56(6):739-745). The interaction between PD-1 and PD-L1 canact as an immune checkpoint, which can lead to, e.g., a decrease intumor infiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and/or immuno evasion by cancerous cells (Dong et al.(2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol.Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res.10:5094-100) Immune suppression can be reversed by inhibiting the localinteraction of PD-1 with PD-L1 or PD-L2; the effect is additive when theinteraction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002)Proc. Nat'l. Acad. Sci USA 99:12293-7; Brown et al. (2003) J. Immunol.170:1257-66).

PD-1 is an inhibitory member of the extended CD28/CTLA-4 family of Tcell regulators. Other members of the CD28 family include CD28, CTLA-4,ICOS and BTLA. PD-1 is suggested to exist as a monomer, lacking theunpaired cysteine residue characteristic of other CD28 family members.PD-1 is expressed on activated B cells, T cells, and monocytes (Okazakiet al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J.Immunol. 170:711-8). Two ligands for PD-1 have been identified, PD-L1(B7-H1) and PD-L2 (B7-DC), that have been shown to downregulate T cellactivation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med.192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). BothPD-L1 and PD-L2 are B7 homologs that bind to PD-1. PD-L1 is abundant ina variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).

The PD-1 gene encodes a 55 kDa type I transmembrane protein that is partof the Ig gene superfamily (Agata et al. (1996) Int Immunol. 8:765-72).The complete PD-1 sequence can be found under GenBank Accession No.U64863. Although structurally similar to CTLA-4, PD-1 lacks the MYPPYmotif (SEQ ID NO: 10) that is important for B7-1 and B7-2 binding.

In view of the above, monoclonal antibodies as PD-1 antagonists havebeen developed in recent years for use in therapy, more precisely fortreating various diseases, including cancer and infectious diseases(e.g., WO2006/121168; WO2015/112900). Any one of such antibodies can beused according to the present invention.

The present inventors have surprisingly found that treating individualswith a combination of Compound A and Compound B as defined above/belowleads to a significant stronger reduction—or even shrinkage—in tumorvolume as compared to treatment with Compound A only or Compound B only.Moreover, when compared to a treatment of individuals with vanucizumaband the same PD-1 antagonist, treatment with a composition comprisingCompound A and Compound B as defined above/below even resulted in tumorshrinkage (Example 1; FIG. 1). Both Compound A and Compound B are activeagents according to the present invention.

A PD-1 antagonist within the meaning of this invention is a compoundthat inhibits the interaction of PD-1 with its receptor(s) or ligand(s).

Preferably, the PD-1 antagonist is an inhibitor of PD-1 or an inhibitorof PD-L1. The PD-1 antagonist may preferably be an anti-PD-1-antibody oran anti-PD-L1-antibody, and more preferably a humanized or fully humananti-PD-1 antibody or a humanized or fully human anti-PD-L1 antibody.Any one of these antibodies may be a recombinant human antibody.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), single chain antibodies, single domainantibodies, and fragmented antibodies (also referred to as antibodyfragments), such as Fab, F(ab)₂, F(ab′)₂, Fab′, single chainvariable-fragments (scFv) or antigen binding domains of an antibody, solong as they exhibit the desired antigen-binding activity.

The antibody may have an effector function, such as ADCC or CDC, that isusually mediated by the Fc part (antibody constant region) of theantibody, or it may have no effector function, e.g. by lacking a Fc partor having a blocked, masked Fc part, in essence a Fc part that is not orinsufficiently recognized by immune cells or immune system components,like the complement system.

The antibody or its fragment may be of any type, e.g. IgA, IgD, IgE,IgG, IgM. Preferred is IgG.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

A “recombinant antibody” is an antibody which has been produced by arecombinantly engineered host cell. It is optionally isolated orpurified.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human cell or derivedfrom a non-human source that utilizes human antibody repertoires orother human antibody-encoding sequences. This definition of a humanantibody specifically excludes a humanized antibody comprising non-humanantigen-binding residues.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human hypervariable regions (HVRs) and amino acidresidues from human FRs. In certain embodiments, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the HVRs (e.g.complementary determining regions (CDRs)) correspond to those of anon-human antibody, and all or substantially the entire frameworkregions (FRs) correspond to those of a human antibody. A humanizedantibody optionally may comprise at least a portion of an antibodyconstant region derived from a human antibody. A “humanized form” of anantibody, e.g. a non-human antibody, refers to an antibody that hasundergone humanization.

“Binding” of a polypeptide (such as an immunoglobulin, an antibody, animmunoglobulin single variable domain of the invention, or generally anantigen-binding molecule or a fragment thereof) means “having affinityfor” or “having specificity for” a certain epitope, antigen or protein(or for at least one part, fragment or epitope thereof).

Generally, the term “specificity” refers to the number of differenttypes of antigens or epitopes to which a particular antigen-bindingmolecule or antigen-binding protein (such as an immunoglobulin singlevariable domain of the invention) molecule can bind. The specificity ofantigen-binding molecule can be determined based on its affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an epitope and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an epitopeand the antigen-binding molecule (alternatively, the affinity can alsobe expressed as the affinity constant (KA), which is 1/KD). As will beclear to the skilled person (for example on the basis of the furtherdisclosure herein), affinity can be determined in a manner known per se,depending on the specific antigen of interest. Avidity is the measure ofthe strength of binding between an antigen-binding molecule (such as animmunoglobulin, an antibody, an immunoglobulin single variable domain ora polypeptide) containing it and the pertinent antigen. Avidity isrelated to both the affinity between an epitope and its antigen bindingsite on the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule.

Natural antibodies, for example, are monospecific. The term“monospecific antibody” as used herein denotes an antibody that has oneor more binding sites each of which bind to the same epitope of the sameantigen. “Multispecific antibodies” bind two or more different epitopes(for example, two, three, four, or more different epitopes). Theepitopes may be on the same or different antigens. An example of amultispecific antibody is a “bispecific antibody” which binds twodifferent epitopes. When an antibody possesses more than onespecificity, the recognized epitopes may be associated with a singleantigen or with more than one antigen.

An epitope is a region of an antigen that is bound by an antibody orantigen binding moiety. The term “epitope” includes any polypeptidedeterminant capable of specific binding to an antibody or antigenbinding moiety. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,glycan side chains, phosphoryl, or sulfonyl, and, in certainembodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. Conformationaland non-conformational epitopes are distinguished in that the binding tothe former but not the latter is lost in the presence of denaturingsolvents. As used herein, the terms “binding” and “specific binding”refer to the binding of the antibody or antigen binding moiety to anepitope of the antigen in an in vitro assay, preferably in a plasmonresonance assay (BIAcore®, GE-Healthcare Uppsala, Sweden) with purifiedwild-type antigen.

The affinity of the binding of the binding molecules (e.g. antibodies)according to the present invention, including antibodies thereof ofCompound A or Compound B, to an antigen is defined by the terms k_(a)(rate constant for the association of the antibody from theantibody/antigen complex), k_(D) (dissociation constant), and K_(D)(kD/ka). In one embodiment binding or that/which specifically binds tomeans a binding affinity (K_(D)) of 10⁻⁸ mol/1 or less, in oneembodiment 10⁻⁸ M to 10⁻¹³ mol/1. Thus, an multispecific antibodyaccording to the invention specifically binds to each antigen for whichit is specific with a binding affinity (K_(D)) of 10⁻⁸ mol/1 or less,e.g. with a binding affinity (K_(D)) of 10⁻⁸ to 10⁻¹³ mol/1. In oneembodiment, with a binding affinity (K_(D)) is 10⁻⁹ to 10⁻¹³ mol/1.

The expressions “variable domains” or “variable region” or Fv as usedherein denotes each of the pair of light and heavy chains which isinvolved directly in binding the antibody to the antigen. The variabledomain of a light chain is abbreviated as “VL” and the variable domainof a heavy chain is abbreviated as “VH”. The variable light and heavychain domains have the same general structure and each domain comprisesfour framework (FR) regions whose sequences are widely conserved,connected by three HVRs (or CDRs). The framework regions adopt abeta-sheet conformation and the CDRs may form loops connecting thebeta-sheet structure. The CDRs in each chain are held in theirthree-dimensional structure by the framework regions and form togetherwith the CDRs from the other chain the antigen binding site. Theantibody's heavy and light chain CDR3 regions play a particularlyimportant role in the binding specificity/affinity of the antibodiesaccording to the invention and therefore provide a further object of theinvention.

The term “constant domains” or “constant region” as used within thecurrent application denotes the sum of the domains of an antibody otherthan the variable region. The constant region is not directly involvedin binding of an antigen, but exhibits various effector functions.

The “constant domains” as used in the antibodies disclosed herein arepreferably from human origin, which is from a constant heavy chainregion of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4and/or a constant light chain kappa or lambda region. Such constantdomains and regions are well known in the state of the art and e.g.described by Kabat et al. (“Sequence of proteins of immunologicalinterest”, US Public Health Services, NIH Bethesda, Md., Publication No.91).

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibits various effector functions. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Accordingto the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ and μ, respectively. The Fc partof an antibody is directly involved in ADCC (antibody-dependentcell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity)based on complement activation, C1q binding and Fc receptor binding.Complement activation (CDC) is initiated by binding of complement factorC1q to the Fc part of most IgG antibody subclasses. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in the Fcpart. Such binding sites are known in the state of the art and describede.g. by Boackle, R. J., et al, Nature 282 (1979) 742-743; Lukas, T. J.,et al, J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al, Nature 288(1980) 338-344; Thommesen, J. E., et al, Mol. Immunol. 37 (2000)995-1004; Idusogie, E. E., et al, J. Immunol. 164 (2000) 4178-4184;Hezareh, M., et al, J. Virology 75 (2001) 12161-12168; Morgan, A., etal, Immunology 86 (1995) 319-324; EP 0 307 434. Such binding sites aree.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numberingaccording to EU index of Kabat, E. A., see below). Antibodies ofsubclass IgG1, IgG2 and IgG3 usually show complement activation and C1qand C3 binding, whereas IgG4 do not activate the complement system anddo not bind C1q and C3.

Regarding Compound A, the term “bispecific binding molecule” refers to amolecule comprising at least one Ang2-binding molecule (or “Ang2-bindingcomponent”) and at least one VEGF-binding molecule (or “VEGF-bindingcomponent”). A bispecific binding molecule may contain more than oneAng2-binding molecule and/or more than one VEGF-binding molecule, i.e.in the case that the bispecific binding molecule contains a biparatopic(as defined below) Ang2-binding molecule and/or a biparatopicVEGF-binding molecule, in part of the molecule that binds to Ang2 or toVEGF, i.e. in its “Ang2-binding component” (or anti-Ang2 component) or“VEGF-binding component” (or anti-VEGF component), respectively. Theword “bispecific” in this context is however not to be construed as toexclude further binding components with binding specificity to moleculesother than VEGF and Ang2 from the bispecific binding molecule.Non-limiting examples of such further binding components are bindingcomponents binding to serum albumin.

Unless indicated otherwise, the term “VEGF-binding molecule” or“Ang2-binding molecule” includes anti-VEGF or anti-Ang2 antibodies,anti-VEGF antibody or anti-Ang2 antibody fragments”, “anti-VEGFantibody-like molecules” or “anti-Ang2 antibody-like molecules”, asdefined herein, and conjugates with any of these. Antibodies include,but are not limited to, monoclonal and chimerized monoclonal antibodies.The term “antibody” encompasses complete immunoglobulins, likemonoclonal antibodies produced by recombinant expression in host cells,as well as antibody fragments or “antibody-like molecules”, includingsingle-chain antibodies and linear antibodies, so-called “SMIPs” (“SmallModular Immunopharmaceuticals”), as e.g. described in WO2002/056910;Antibody-like molecules include immunoglobulin single variable domains,as defined herein. Other examples for antibody-like molecules areimmunoglobulin super family antibodies (IgSF), or CDR-grafted molecules.

“Ang2-binding molecule” or “VEGF-binding molecule” respectively, refersto both monovalent target-binding molecules (i.e. molecules that bind toone epitope of the respective target) as well as to bi- or multivalentbinding molecules (i.e. binding molecules that bind to more than oneepitope, e.g. “biparatopic” molecules as defined hereinbelow). Ang2 (orVEGF)-binding molecules containing more than one Ang2 (or VEGF)-bindingimmunoglobulin single variable domain are also termed “formatted”binding molecules, they may, within the target-binding component, inaddition to the immunoglobulin single variable domains, comprise linkersand/or moieties with effector functions, e.g. half-life-extendingmoieties like albumin-binding immunoglobulin single variable domains,and/or a fusion partner like serum albumin and/or an attached polymerlike PEG.

The term “biparatopic Ang2 (or VEGF)-binding molecule” or “biparatopicimmunoglobulin single variable domain” as used herein shall mean abinding molecule comprising a first immunoglobulin single variabledomain and a second immunoglobulin single variable domain as definedherein, wherein the two molecules bind to two non-overlapping epitopesof the respective antigen. The biparatopic binding molecules arecomposed of immunoglobulin single variable domains which have differentspecificities with respect to the epitope.

A formatted binding molecule may, albeit less preferred, also comprisetwo identical immunoglobulin single variable domains or two differentimmunoglobulin single variable domains that recognize the same oroverlapping epitopes or their respective antigen. In this case, withrespect to VEGF, the two immunoglobulin single variable domains may bindto the same or an overlapping epitope in each of the two monomers thatform the VEGF dimer.

The efficacy of the bispecific binding molecule of the invention, and ofcompositions comprising the same, can be tested using any suitable invitro assay, cell-based assay, in vivo assay and/or animal model knownper se, or any combination thereof, depending on the specific disease ordisorder of interest. Suitable assays and animal models will be clear tothe skilled person, and for example include the assays described in EP 2694 546 B1.

Unless indicated otherwise, the terms “immunoglobulin” and“immunoglobulin sequence”—whether used herein to refer to a heavy chainantibody or to a conventional 4-chain antibody—are used as general termsto include both the full-size antibody, the individual chains thereof,as well as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as VHH domains orVH/VL domains, respectively). In addition, the term “sequence” as usedherein (for example in terms like “immunoglobulin sequence”, “antibodysequence”, “(single) variable domain sequence”, “VHH sequence” or“protein sequence”) should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation.

The term “domain” (of a polypeptide or protein) as used herein refers toa folded protein structure which has the ability to retain its tertiarystructure independently of the rest of the protein. Generally, domainsare responsible for discrete functional properties of proteins, and inmany cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g. a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin theimmunoglobulin fold characteristic of antibody molecules, which consistsof a 2-layer sandwich of about 7 antiparallel beta-strands arranged intwo beta-sheets, optionally stabilized by a conserved disulphide bond.An immunoglobulin domain comprises (a) variable domain(s), i.e. one ormore immunoglobulin variable domains.

The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and hereinbelow as “framework region 1”or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or“FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementary determiningregions” or “CDRs”, which are referred to in the art and hereinbelow as“complementary determining region 1” or “CDR1”; as “complementarydetermining region 2” or “CDR2”; and as “complementary determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site. The molecules of thepresent invention include immunoglobulin single variable domains likeVHHs.

The term “immunoglobulin single variable domain” as used herein means animmunoglobulin variable domain which is capable of specifically bindingto an epitope of the antigen without pairing with an additional variableimmunoglobulin domain. One example of immunoglobulin single variabledomains in the meaning of the present invention are “domain antibodies”,such as the immunoglobulin single variable domains VH and VL (VH domainsand VL domains). Another example of immunoglobulin single variabledomains are “VHH domains” (or simply “VHHs”) from camelids, as describedhereinafter.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e. by a VH-VL pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

“VHH domains”, also known as VHHs, VHH domains, VHH antibody fragments,and VHH antibodies, have originally been described as the antigenbinding immunoglobulin (variable) domain of “heavy chain antibodies”(i.e. of “antibodies devoid of light chains”; Hamers-Casterman C,Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa E B, BendahmanN, Hamers R.: “Naturally occurring antibodies devoid of light chains”;Nature 363, 446-448 (1993)). The term “VHH domain” has been chosen inorder to distinguish these variable domains from the heavy chainvariable domains that are present in conventional 4-chain antibodies(which are referred to herein as “V_(H) domains” or “VH domains”) andfrom the light chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “V_(L) domains” or“VL domains”). VHH domains can specifically bind to an epitope withoutan additional antigen binding domain (as opposed to VH or VL domains ina conventional 4-chain antibody, in which case the epitope is recognizedby a VL domain together with a VH domain). VHH domains are small, robustand efficient antigen recognition units formed by a singleimmunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH,V_(H)H domain, VHH antibody fragment, VHH antibody are usedinterchangeably and are representatives of immunoglobulin singlevariable domains (having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4and specifically binding to an epitope without requiring the presence ofa second immunoglobulin variable domain), and which are distinguishedfrom VH domains by the so-called “hallmark residues”, as defined in e.g.WO 2009/109635, FIG. 1.

The amino acid residues of a immunoglobulin single variable domain, e.g.a VHH, are numbered according to the general numbering for V_(H) domainsgiven by Kabat et al. (“Sequence of proteins of immunological interest”,US Public Health Services, NIH Bethesda, Md., Publication No. 91), asapplied to VHH domains from Camelids, as shown e.g. in FIG. 2 ofRiechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).According to this numbering

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V_(H)domains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain one or moreamino acid residues than the number allowed for by the Kabat numbering).This means that, generally, the numbering according to Kabat may or maynot correspond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and figures, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Immunoglobulin single variable domains, e.g. VHHs and domain antibodies,according to the preferred embodiments of the invention, have a numberof unique structural characteristics and functional properties whichmakes them highly advantageous for use in therapy as functionalantigen-binding molecules. In particular, and without being limitedthereto, VHH domains (which have been “designed” by nature tofunctionally bind to an antigen without pairing with a light chainvariable domain) can function as single, relatively small, functionalantigen-binding structural units.

Further details and information about advantages of immunoglobulinsingle variable domains and about obtaining VHHs are disclosed in detailin EP 2 694 546 B1.

The immunoglobulin single variable domains of the invention are notlimited with respect to a specific biological source from which theyhave been obtained or to a specific method of preparation. Suitablemethods and techniques for obtaining VHH domains binding to a specificantigen or epitope have been described in WO2006/040153 andWO2006/122786.

According to specific embodiments, the immunoglobulin single variabledomains of the invention are VHH domains with an amino acid sequencethat essentially corresponds to the amino acid sequence of a naturallyoccurring VHH domain, but that has been “humanized” or“sequence-optimized” (optionally after affinity-maturation), i.e. byreplacing one or more amino acid residues in the amino acid sequence ofsaid naturally occurring VHH sequence by one or more of the amino acidresidues that occur at the corresponding position(s) in a variable heavydomain of a conventional 4-chain antibody from a human being. This canbe performed using methods known in the art, which can be routinely usedby the skilled person.

A humanized VHH domain may contain one or more fully human frameworkregion sequences, and, in an even more specific embodiment, may containhuman framework region sequences derived from the human germline Vh3sequences DP-29, DP-47, DP-51, or parts thereof, or be highly homologousthereto, optionally combined with JH sequences, such as JH5. Thus, ahumanization protocol may comprise the replacement of any of the VHHresidues with the corresponding framework 1, 2 and 3 (FR1, FR2 and FR3)residues of germline VH genes such as DP 47, DP 29 and DP 51) eitheralone or in combination. Suitable framework regions (FR) of theimmunoglobulin single variable domains of the invention can be selectedfrom those as set out e.g. in WO 2006/004678 and specifically, includethe so-called “KERE” and “GLEW” classes. Examples are immunoglobulinsingle variable domains having the amino acid sequence G-L-E-W at aboutpositions 44 to 47, and their respective humanized counterparts. Ahumanized VHH domain may contain one or more fully human frameworkregion sequences.

By way of example, a humanizing substitution for VHHs belonging to the103 P,R,S-group and/or the GLEW-group (as defined below) is 108Q to108L. Methods for humanizing immunoglobulin single variable domains areknown in the art.

Binding immunoglobulin single variable domains with improved propertiesin view of therapeutic application, e g enhanced affinity or decreasedimmunogenicity, may be obtained from individual binding molecules bytechniques known in the art, such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, humanizing, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing, also termed “sequence optimization”, as described herein.Reference is, for example, made to standard handbooks, as well as to thefurther description and Examples.

In accordance with the above, in a preferred embodiment, theimmunoglobulin single variable domains of Compound A are VHH domains.More preferably, Compound A is selected from compounds having thefollowing amino acid sequences:

(SEQ ID NO: 11) DVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTIS RDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAK TTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSC AVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTA VYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS; (SEQ ID NO: 12) EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTIS RDNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVE SGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAK TTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSC AVSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTA VYYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS; and (SEQ ID NO: 13) VQLVESGGGLVQPGGSLRLSCAASGRTFSSYSMGWFRQAPGKEREFVVAISKGGYKYDAVSLEGRFTISR DNAKNTVYLQINSLRPEDTAVYYCASSRAYGSSRLRLADTYEYWGQGTLVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKT TLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCA VSGITLDDYAIGWFRQAPGKEREGVSAIRSSGGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRPEDTAV YYCAAVPAGRLRYGEQWYPIYEYDAWGQGTLVTVSS.

The three sequences shown above are almost identical, except for theN-terminal sequence, which can be changed or modified in order tooptimally adapt the sequence to the selected expression system(expression vector, host cell) or because of other needs. Thus, it isclear to the skilled person that further polypeptides which essentiallycomprise the above sequence, but which are slightly modified, e.g. byamino acid exchanges, deletions or additions which do not change thebinding affinities of the resulting polypeptides, will also be useful asa “Compound A” of a pharmaceutical combination according to theinvention.

In another embodiment, the representatives of the class of VEGF- and/orAng2-binding immunoglobulin single variable domains of the inventionhave amino acid sequences that correspond to the amino acid sequence ofa naturally occurring VH domain that has been “camelized”, i.e. byreplacing one or more amino acid residues in the amino acid sequence ofa naturally occurring variable heavy chain from a conventional 4-chainantibody by one or more amino acid residues that occur at thecorresponding position(s) in a VHH domain of a heavy chain antibody.This can be performed in a manner known per se, which will be clear tothe skilled person, and reference is additionally be made toWO1994/04678. Such camelization may preferentially occur at amino acidpositions which are present at the VH-VL interface and at the so-calledCamelidae Hallmark residues (see for example also WO1994/04678). Adetailed description of such “humanization” and “camelization”techniques and preferred framework region sequences consistent therewithcan additionally be taken from e.g. pp. 98 of WO2006/040153 and pp. 107of WO2006/122786.

A PD-1 antagonist within the meaning of this invention and all of itsembodiments is a compound that inhibits the interaction of PD-1 with itsreceptor(s). PD-1 antagonists are well-known in the art, e.g. reviewedby Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein byreference). Any PD-1 antagonist, especially antibodies, such as thosedisclosed by Li et al. as well as the further antibodies disclosedherein below, can be used according to the invention. Preferably, thePD-1 antagonist of this invention and all its embodiments is selectedfrom the group consisting of the following antibodies:

-   -   pembrolizumab (anti-PD-1 antibody);    -   nivolumab (anti-PD-1 antibody);    -   pidilizumab (anti-PD-1 antibody);    -   PDR-001 (anti-PD-1 antibody);    -   PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 as disclosed herein below        (anti-PD-1 antibodies)    -   atezolizumab (anti-PD-L1 antibody);    -   avelumab (anti-PD-L1 antibody);    -   durvalumab (anti-PD-L1 antibody).

Pembrolizumab (formerly also known as lambrolizumab; trade nameKeytruda; also known as MK-3475) disclosed e.g. in Hamid, O. et al.(2013) New England Journal of Medicine 369(2):134-44, is a humanizedIgG4 monoclonal antibody that binds to PD-1; it contains a mutation atC228P designed to prevent Fc-mediated cytotoxicity. Pembrolizumab ise.g. disclosed in U.S. Pat. No. 8,354,509 and WO2009/114335. It isapproved by the FDA for the treatment of patients suffering fromunresectable or metastatic melanoma and patients with metastatic NSCLC.

Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) isa fully human IgG4 monoclonal antibody which specifically blocks PD-1,lacking detectable antibody-dependent cellular toxicity (ADCC).Nivolumab is e.g. disclosed in U.S. Pat. No. 8,008,449 andWO2006/121168. It has been approved by the FDA for the treatment ofpatients suffering from unresectable or metastatic melanoma, metastaticNSCLC and advanced renal cell carcinoma.

Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibodythat binds to PD-1. Pidilizumab is e.g. disclosed in WO 2009/101611.

PDR-001 or PDR001 is a high-affinity, ligand-blocking, humanizedanti-PD-1 IgG4 antibody that blocks the binding of PD-L1 and PD-L2 toPD-1. PDR-001 is disclosed in WO2015/112900 and WO2017/019896.

Antibodies PD1-1 to PD1-5 are antibody molecules defined by thesequences as shown in Table 1, wherein HC denotes the (full length)heavy chain and LC denotes the (full length) light chain:

TABLE 1 SEQ ID Sequence NO: name Amino acid sequence 14 HC ofEVMLVESGGGLVQPGGSLRL PD1-1 SCTASGFTFSASAMSWVRQA PGKGLEWVAYISGGGGDTYYSSSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARHS NVNYYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG 15 LC of EIVLTQSPATLSLSPGERAT PD1-1MSCRASENIDTSGISFMNWY QQKPGQAPKLLIYVASNQGS GIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQSKEVPW TFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 16 HC of EVMLVESGGGLVQPGGSLRL PD1-2SCTASGFTFSASAMSWVRQA PGKGLEWVAYISGGGGDTYY SSSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHS NPNYYAMDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLG 17LC of EIVLTQSPATLSLSPGERAT PD1-2 MSCRASENIDTSGISFMNWYQQKPGQAPKLLIYVASNQGS GIPARFSGSGSGTDFTLTIS RLEPEDFAVYYCQQSKEVPWTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 18 HC ofEVMLVESGGGLVQPGGSLRL PD1-3 SCTASGFTFSKSAMSWVRQA PGKGLEWVAYISGGGGDTYYSSSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARHS NVNYYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG 19 LC of EIVLTQSPATLSLSPGERAT PD1-3MSCRASENIDVSGISFMNWY QQKPGQAPKLLIYVASNQGS GIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQSKEVPW TFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 HC of EVMLVESGGGLVQPGGSLRL PD1-4SCTASGFTFSKSAMSWVRQA PGKGLEWVAYISGGGGDTYY SSSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHS NVNYYAMDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLG 21LC of EIVLTQSPATLSLSPGERAT PD1-4 MSCRASENIDVSGISFMNWYQQKPGQAPKLLIYVASNQGS GIPARFSGSGSGTDFTLTIS RLEPEDFAVYYCQQSKEVPWTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 22 HC ofEVMLVESGGGLVQPGGSLRL PD1-5 SCTASGFTFSKSAMSWVRQA PGKGLEWVAYISGGGGDTYYSSSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARHS NVNYYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG 23 LC of EIVLTQSPATLSLSPGERAT PD1-5MSCRASENIDVSGISFMNWY QQKPGQAPKLLIYVASNQGS GIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQSKEVPW TFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Specifically, the anti-PD-1 antibody molecule described herein abovehas:

(PD1-1:) a heavy chain comprising the amino acid sequence of SEQ ID NO:14 and a light chain comprising the amino acid sequence of SEQ ID NO:15; or(PD1-2:) a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and a light chain comprising the amino acid sequence of SEQ ID NO:17; or(PD1-3:) a heavy chain comprising the amino acid sequence of SEQ ID NO:18 and a light chain comprising the amino acid sequence of SEQ ID NO:19; or(PD1-4:) a heavy chain comprising the amino acid sequence of SEQ ID NO:20 and a light chain comprising the amino acid sequence of SEQ ID NO:21; or(PD1-5:) a heavy chain comprising the amino acid sequence of SEQ ID NO:22 and a light chain comprising the amino acid sequence of SEQ ID NO:23.

Atezolizumab (Tecentriq, also known as MPDL3280A) is a phage-derivedhuman IgG1k monoclonal antibody targeting PD-L1 and is described e.g. inDeng et al. mAbs 2016; 8:593-603. It has been approved by the FDA forthe treatment of patients suffering from urothelial carcinoma.

Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody anddescribed in e.g. Boyerinas et al. Cancer Immunol. Res. 2015;3:1148-1157.

Durvalumab (MEDI4736) is a human IgG1k monoclonal antibody with highspecificity to PD-L1 and described in e.g. Stewart et al. CancerImmunol. Res. 2015; 3:1052-1062 or in Ibrahim et al. Semin Oncol. 2015;42:474-483.

Further PD-1 antagonists disclosed by Li et al. (supra), or known to bein clinical trials, such as AMP-224, MEDI0680 (AMP-514), REGN2810,BMS-936559, JS001-PD-1, SHR-1210, BMS-936559, TSR-042, JNJ-63723283,MEDI4736, MPDL3280A, and MSB0010718C, may be used as alternative or inaddition to the above mentioned antagonists.

The INNs as used herein are meant to also encompass all biosimilarantibodies having the same, or substantially the same, amino acidsequences as the originator antibody, including but not limited to thosebiosimilar antibodies authorized under 42 USC § 262 subsection (k) inthe US and equivalent regulations in other jurisdictions.

PD-1 antagonists listed above are known in the art with their respectivemanufacture, therapeutic use and properties.

In one embodiment the PD-1 antagonist is pembrolizumab.

In another embodiment the PD-1 antagonist is nivolumab.

In another embodiment the PD-1 antagonist is pidilizumab.

In another embodiment the PD-1 antagonist is atezolizumab.

In another embodiment the PD-1 antagonist is avelumab.

In another embodiment the PD-1 antagonist is durvalumab.

In another embodiment the PD-1 antagonist is PDR-001.

In another embodiment the PD-1 antagonist is PD1-1.

In another embodiment the PD-1 antagonist is PD1-2.

In another embodiment the PD-1 antagonist is PD1-3.

In another embodiment the PD-1 antagonist is PD1-4.

In another embodiment the PD-1 antagonist is PD1-5.

Within this invention it is to be understood that the combinations,compositions, kits, methods, uses or compounds for use according to thisinvention may envisage the simultaneous, concurrent, sequential,successive, alternate or separate administration of the active agents orcomponents. It will be appreciated that the bispecific binding moleculeand the PD-1 antagonist can be administered formulated eitherdependently or independently, such as e.g. the bispecific bindingmolecule and the PD-1 antagonist may be administered either as part ofthe same pharmaceutical composition/dosage form or, preferably, inseparate pharmaceutical compositions/dosage forms.

In this context, “combination” or “combined” within the meaning of thisinvention includes, without being limited, a product that results fromthe mixing or combining of more than one active agent and includes bothfixed and non-fixed (e.g. free) combinations (including kits) and uses,such as e.g. the simultaneous, concurrent, sequential, successive,alternate or separate use of the components or agents. The term “fixedcombination” means that the active agents are both administered to apatient simultaneously in the form of a single entity or dosage. Theterm “non-fixed combination” means that the active agents are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeagents.

The administration of the bispecific binding molecule/Compound A and thePD-1 antagonist/Compound B may take place by co-administering the activecomponents or agents, such as e.g. by administering them simultaneouslyor concurrently in one single or in two separate formulations or dosageforms. Alternatively, the administration of the bispecific bindingmolecule and the PD-1 antagonist may take place by administering theactive components or agents sequentially or in alteration, such as e.g.in two separate formulations or dosage forms.

For example, simultaneous administration includes administration atsubstantially the same time. This form of administration may also bereferred to as “concomitant” administration. Concurrent administrationincludes administering the active agents within the same general timeperiod, for example on the same day(s) but not necessarily at the sametime. Alternate administration includes administration of one agentduring a time period, for example over the course of a few days or aweek, followed by administration of the other agent during a subsequentperiod of time, for example over the course of a few days or a week, andthen repeating the pattern for one or more cycles. Sequential orsuccessive administration includes administration of one agent during afirst time period (for example over the course of a few days or a week)using one or more doses, followed by administration of the other agentduring a second time period (for example over the course of a few daysor a week) using one or more doses. An overlapping schedule may also beemployed, which includes administration of the active agents ondifferent days over the treatment period, not necessarily according to aregular sequence. Variations on these general guidelines may also beemployed, e.g. according to the agents used and the condition of thesubject.

Accordingly, in a preferred embodiment, in the method according thepresent invention, Compound A as described herein is administeredsimultaneously, concurrently, sequentially, successively, alternately orseparately with Compound B as described herein. In a similar preferredembodiment, Compound A as described herein for use in a method accordingto the present invention, is administered simultaneously, concurrently,sequentially, successively, alternately or separately with Compound B asdescribed herein. In a related preferred embodiment, Compound B, asdescribed herein for use in a method according to the present invention,is administered simultaneously, concurrently, sequentially,successively, alternately or separately with Compound A as describedherein. In a further preferred embodiment, the use of Compound A asdescribed herein is provided wherein Compound A is to be administeredsimultaneously, concurrently, sequentially, successively, alternately orseparately with Compound B. In a further related preferred embodiment,the use of Compound B as described herein is provided wherein Compound Bis to be administered simultaneously, concurrently, sequentially,successively, alternately or separately with Compound A. In anotherembodiment, the kit according to the present invention is providedwherein the first pharmaceutical composition is to be administeredsimultaneously, concurrently, sequentially, successively, alternately orseparately with the second pharmaceutical composition.

Preferred routes of administration for Compound A, Compound B, or both,administered separately or simultaneously, include, but are not limitedto, oral, enterical, parenteral (e.g. intramuscular, intraperitoneal,intravenous, transdermal or subcutaneous injection, or implant), nasal,vaginal, rectal, or topical administration. In a preferred embodiment,the route of administration is intravenous administration, especiallyintravenous infusion or injection. The compounds of the presentinvention may be formulated, alone or together, in suitable dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, excipients and/or vehicles appropriate for eachroute of administration. More preferably, formulations include solid,semi-solid or liquid dosage forms, such as lyophilisation, liquidsolutions (e.g. injectable and infusible solutions), dispersions orsuspensions, liposomes and suppositories. The preferred mode depends onthe intended mode of administration and therapeutic application.Especially preferred embodiments include liquid formulations andlyophilisation. In the case of a lyophilisation, the lyophilisate may bereconstituted in a liquid, preferably water.

The compounds as described herein may be administered daily, 5 times aweek, 3 times a week, 2 times a week, once a week, once in 2 weeks, oncein 3 weeks, once in 4 weeks. Preferable administration intervals includeonce a week and once in 2 weeks.

Preferably, Compounds A and B are administered once a week by i.v.infusion.

An administration regimen may include long-term treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months or years of duration. Necessary modifications in this dosageregimen may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication. Administration may be daily, everysecond day, every third day, every fourth day, one day per week, twodays per week, one day per two weeks, one day per three weeks, etc.

The compounds as described herein may be administered at therapeuticallyeffective amounts in single or divided doses administered at appropriatetime intervals. A therapeutically effective amount refers to an amounteffective at dosages and for periods of time necessary to achieve thedesired therapeutic result and is the minimum amount necessary toprevent, ameliorate, or treat a disease or disorder. A therapeuticallyeffective amount of the compounds according to the present invention mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the compound to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the compound isoutweighed by the therapeutically beneficial effects. A therapeuticallyeffective dose preferably inhibits a measurable parameter, e.g. a tumorgrowth rate by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects orrelative to a preceding untreated period of the same subject that is tobe treated.

The active compounds may be administered in such doses which aretherapeutically effective in monotherapy, or in such doses which arelower or higher than the doses used in monotherapy, but when combinedresult in a desired (jointly) therapeutically effective amount. Theamount of the bispecific binding molecules of the invention required foruse in treatment may be adapted to the particular binding moleculeselected, the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also, thedosage of the binding molecules of the invention may be adapteddepending on the target cell, tumor, tissue, graft, or organ.

The desired dose of Compound A or Compound B may be administered as afixed amount per administration or as bolus, to reach a set bloodconcentration in the patient.

Administration of Compound B, the PD-1 antagonist, as described hereinmay e.g. be by injection (e.g. subcutaneously or intravenously) at adose of about 0.1 to 30 mg/kg of patient body weight, e.g. about 0.5 to25 mg/kg of patient body weight, about 1 to 20 mg/kg of patient bodyweight, about 2 to 5 mg/kg of patient body weight, or about 3 mg/kg ofpatient body weight.

Dosages and therapeutic regimens of the PD-1 antagonist can bedetermined by a skilled artisan. Preferred dosage regimens for a PD-1antagonist of the invention include 1 mg/kg of host body weight or 3mg/kg of host body weight via intravenous administration, with theantibody being given using one of the following dosing schedules: (i)every four weeks for six dosages, then every three months; (ii) everythree weeks; (iii) 3 mg/kg of host body weight once followed by 1 mg/kgof host body weight every three weeks. In certain embodiments, the PD-1antagonist is administered by injection (e.g., subcutaneously orintravenously) at a dose of about 1 to 40 mg/kg of host body weight,e.g., 1 to 30 mg/kg of host body weight, e.g., about 5 to 25 mg/kg ofhost body weight, about 10 to 20 mg/kg of host body weight, about 1 to 5mg/kg of host body weight, 1 to 10 mg/kg of host body weight, 5 to 15mg/kg of host body weight, 10 to 20 mg/kg of host body weight, 15 to 25mg/kg of host body weight, or about 3 mg/kg of host body weight. Thedosing schedule can vary from e.g., once a week to once every 2, 3, or 4weeks. In one embodiment, the PD-1 antagonist is administered at a dosefrom about 10 to 20 mg/kg of host body weight every other week. Theantibody molecule can be administered by intravenous infusion at a rateof more than 20 mg/min, e.g., 20-40 mg/min, and typically greater thanor equal to 40 mg/min to reach a dose of about 35 to 440 mg/m²,typically about 70 to 310 mg/m², and more typically, about 110 to 130mg/m². In embodiments, the infusion rate of about 110 to 130 mg/m²achieves a level of about 3 mg/kg of host body weight. In otherembodiments, the antibody molecule can be administered by intravenousinfusion at a rate of less than 10 mg/min, e.g., less than or equal to 5mg/min to reach a dose of about 1 to 100 mg/m², e.g., about 5 to 50mg/m², about 7 to 25 mg/m², or, about 10 mg/m². In some embodiments, theantibody is infused over a period of about 30 min. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The dosing schedule of Compound A and Compound B, separately ortogether, may vary from e.g. once a week to once every 2, 3 or 4 weeks.In a certain embodiment, the administered amount or dosage of CompoundA, Compound B, or both, is lower (e.g. at least 20%, at least 30%, atleast 40%, or at least 50% lower). In other embodiments, the amount ordosage of Compound A, Compound B, or both, that results in a desiredeffect (e.g. treatment of a hyperproliferative or oncological disease)is lower (e.g. at least 20%, at least 30%, at least 40%, or at least 50%lower).

The method, compounds, compounds for use, uses of compounds,pharmaceutical composition and kit according to the present inventioncomprises administering to the subject a combination of a bispecificantibody molecule and an anti-PD-1 antibody molecule as describedherein.

Depending on the cancerous disease to be treated, the combinationtherapy as defined herein may be used on its own or in furthercombination with one or more additional therapeutic agents, inparticular selected from chemotherapeutic agents or therapeuticallyactive compounds that inhibit angiogenesis, signal transduction pathwaysor mitotic checkpoints in cancer cells.

The additional therapeutic agent may be administered simultaneouslywith, optionally as a component of the same pharmaceutical preparation,or before or after administration of the binding molecule and/or the PD1antagonist.

The chemotherapeutic agent may be selected from hormones, hormonalanalogues and antihormonals (e.g. tamoxifen, toremifene, raloxifene,fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide,cyproterone acetate, finasteride, buserelin acetate, fludrocortisone,fluoxymesterone, medroxyprogesterone, octreotide, arzoxifene,pasireotide, vapreotide), aromatase inhibitors (e.g. anastrozole,letrozole, liarozole, exemestane, atamestane, formestane), LHRH agonistsand antagonists (e.g. goserelin acetate, leuprolide, abarelix,cetrorelix, deslorelin, histrelin, triptorelin), antimetabolites (e.g.antifolates like methotrexate, pemetrexed, pyrimidine analogues like 5fluorouracil, capecitabine, decitabine, nelarabine, and gemcitabine,purine and adenosine analogues such as mercaptopurine thioguanine,cladribine and pentostatin, cytarabine, fludarabine); antitumorantibiotics (e.g. anthracyclines like doxorubicin, daunorubicin,epirubicin and idarubicin, mitomycin-C, bleomycin dactinomycin,plicamycin, mitoxantrone, pixantrone, streptozocin); platinumderivatives (e.g. cisplatin, oxaliplatin, carboplatin, lobaplatin,satraplatin); alkylating agents (e.g. estramustine, mechlorethamine,melphalan, chlorambucil, busulphan, dacarbazine, cyclophosphamide,ifosfamide, hydroxyurea, temozolomide, nitrosoureas such as carmustineand lomustine, thiotepa); antimitotic agents (e.g. vinca alkaloids likevinblastine, vindesine, vinorelbine, vinflunine and vincristine; andtaxanes like paclitaxel, docetaxel and their formulations, larotaxel;simotaxel, and epothilones like ixabepilone, patupilone, ZK-EPO);topoisomerase inhibitors (e.g. epipodophyllotoxins like etoposide andetopophos, teniposide, amsacrine, topotecan, irinotecan) andmiscellaneous chemotherapeutics such as amifostine, anagrelide,interferon alpha, procarbazine, mitotane, and porfimer, bexarotene,celecoxib.

In a preferred embodiment, the treatment involving Compound A andCompound B further includes a “platinum doublet” therapy, i.e. therapywith (i) a platinum compound such as cisplatin or carboplatin, plus (ii)a third-generation chemotherapy agent such as docetaxel, paclitaxel,vinorelbine, or gemcitabine.

In another preferred embodiment, the treatment involving Compound A andCompound B is combined with a cancer cell targeting therapy.

In certain embodiments, the oncological or hyperproliferative disease,in particular cancer or a tumor disease, treated with the combinationtherapy as disclosed herein, includes but is not limited to, a solidtumor, a hematological cancer (e.g. leukemia, lymphoma, myeloma, e.g.multiple myeloma), and a metastatic lesion. In one embodiment, thecancer is a solid tumor. Examples of solid tumors include malignancies,e.g. sarcomas and carcinomas, e.g. adenocarcinomas of the various organsystems, such as those affecting the lung, breast, ovarian, lymphoid,gastrointestinal (e.g. colon), anal, genitals and genitourinary tract(e.g. renal, urothelial, bladder cells, prostate), pharynx, CNS (e.g.brain, neural or glial cells), head and neck, skin (e.g. melanoma) andpancreas, as well as adenocarcinomas which include malignancies such ascolon cancers, rectal cancer, renal-cell carcinoma, liver cancer,non-small cell lung cancer, cancer of the small intestine and cancer ofthe esophagus. The cancer may be at an early, intermediate, late stageor metastatic cancer.

As used herein, “hyperproliferative disease” refers to conditionswherein cell growth is increased over normal levels. For example,hyperproliferative diseases or disorders include malignant diseases(e.g. esophageal cancer, colon cancer, biliary cancer) and non-malignantdiseases (e.g. atherosclerosis, benign hyperplasia, benign prostatichypertrophy).

In one embodiment, the cancer is chosen from a lung cancer (e.g. NSCLC(e.g. a NSCLC with squamous and/or non-squamous histology, or a NSCLCadenocarcinoma)), a melanoma (e.g. an advanced melanoma), a renal cancer(e.g. a renal cell carcinoma), a liver cancer, a myeloma (e.g. amultiple myeloma), a prostate cancer, a breast cancer (e.g. a breastcancer that does not express one, two or all of estrogen receptor,progesterone receptor, or Her2/neu, e.g. a triple negative breastcancer), a colorectal cancer, a pancreatic cancer, a head and neckcancer (e.g. head and neck squamous cell carcinoma (HNSCC), anal cancer,gastro-esophageal cancer, thyroid cancer, cervical cancer, alymphoproliferative disease (e.g. a post-transplant lymphoproliferativedisease) or a hematological cancer, T-cell lymphoma, B-cell lymphoma, anon-Hodgkin lymphoma, or a leukemia (e.g. a myeloid leukemia or alymphoid leukemia).

In another embodiment, the cancer is chosen from a carcinoma (e.g.advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g. aNSCLC.

In one embodiment, the cancer is a lung cancer, e.g. a NSCLC or smallcell lung cancer. In a preferred embodiment, the cancer is NSCLC.

In an especially preferred embodiment combinable with any aspect andembodiment of the invention, the combination therapy according to thepresent invention is for treating a patient suffering from locallyadvanced or metastatic non-squamous NSCLC without EGFR mutation or ALKtranslocation. Therein, optionally, the patient is selected according tohis/her PD-L1 expression status, such as high or low PD-L1 expressionstatus. The treatment may be a first line treatment, i.e. the firsttreatment given for the disease (NSCLC). Accordingly the patient may nothave undergone chemotherapy or radiation therapy, in particular not forthe treatment of NSCLC when treated according to the invention. Bydetermining the PD-L1 expression status prior to any cancer treatment ofthe patient, reliable results can be achieved.

PD-L1 expression status can be determined as described by Han et al.(Journal of Pathology and Translational Medicine 2017; 51: 40-48) or byWang et al. (OncoTargets and Therapy 2016:9 5023-5039), such as by usingimmunohistochemistry. A biopsy sample of the tumor of the patient to betreated may be used for PD-L1 expression status determination. A singletumor biopsy or multiple biopsies from an individual patient may beused, in case of more than one biopsies used, preferably the biopsy withthe highest PD-L1 expression is used to determined PD-L1 expressionstatus according to the invention. PD-L1 can be determined on cellsurface (membranous) or cytoplasmic expression, expression by tumorcells only and/or by other cells in the tumor milieu (e.g. immunecells). The thresholds for considering PD-L1 positive expression can bee.g. at least 1%, at least 5% or at least 10% PD-L1 expression in cells,e.g. tumor and/or immune cells, wherein samples with ≥1%, ≥5% or ≥10%PD-L1 expression in cells, e.g. as determined by immunohistochemistrycan be considered PD-L1 “positive”. In a preferred embodiment, the PD-L1expression status is determined on tumor cells or immune cells, morepreferably on tumor cells, wherein a PD-L1 expression of 1% to 5% ofcells is considered low PD-L1 expression and a PD-L1 expression of >5%of cells, e.g. >5% to 40% of cells, is considered as high PD-L1expression, as above preferably by determination byimmunohistochemistry, e.g. as described by Han et al. or Wang et al..Patients to be treated may be patients with low PD-L1 expression,patients with high PD-L1 expression, patients with low PD-L1 expressionexcluding patients with high PD-L1 expression or patients with highPD-L1 expression excluding patients with low PD-L1 expression.

In one embodiment, the cancer is a melanoma, e.g. an advanced melanoma.In one embodiment, the cancer is an advanced or unresectable melanomathat does not respond to other therapies. In other embodiments, thecancer is a melanoma with a BRAF mutation (e.g. a BRAF V600 mutation).

In another embodiment, the cancer is a hepatocarcinoma, e.g. an advancedhepatocarcinoma, with or without a viral infection, e.g. a chronic viralhepatitis.

In another embodiment, the cancer is a prostate cancer, e.g. an advancedprostate cancer.

In yet another embodiment, the cancer is a myeloma, e.g. multiplemyeloma.

In yet another embodiment, the cancer is a renal cancer, e.g. a renalcell carcinoma (RCC) (e.g. a metastatic RCC or clear renal cellcarcinoma (CCRCC)).

As outlined above, the present invention relates to a pharmaceuticalcomposition comprising Compound A and Compound B as defined herein andto a kit comprising a first pharmaceutical composition comprisingCompound A as defined herein and to a second pharmaceutical compositioncomprising Compound B as defined herein.

The term “pharmaceutical composition” as defined herein refers to apreparation which is in such form as to permit the biological activityof an active ingredient contained therein to be effective, and whichcontains no additional components which are unacceptably toxic to asubject to which the composition would be administered. A pharmaceuticalcomposition of the present invention can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results.

Regardless of the route of administration selected, the Compounds usedin a combination therapy of the present invention and/or thepharmaceutical composition, the first pharmaceutical composition and thesecond pharmaceutical composition of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

The kit as defined herein may comprise a suitable container or severalsuitable containers comprising the first pharmaceutical compositionand/or the second pharmaceutical composition, wherein the first and thesecond pharmaceutical composition may be contained in the same containeror in different containers. The kit may be used in any method or anyuses of the invention.

Preferably, the kit according to the present invention further comprisesa package insert comprising readable instructions for using Compound Aand/or Compound B in the treatment and/or prevention of an oncologicalor hyperproliferative disease, preferably cancer or a tumor disease,especially NSCLC, in a patient in need thereof. The instructions mayprovide further detailed as described above with regard to the inventivemethod and any of its preferred embodiments.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one”, butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

“About” as used herein means an acceptable degree of error for thequantity measured given the nature of precision of the measurements.Exemplary degrees of error are within 20%, typically within 10%, andmore typically within 5% of a given value or range of values.

The term “treating” or “treatment” as used herein means to cure analready present disease state or condition or to increase the likelihoodof recovery from the disease state or condition. Treating can alsoinclude inhibiting, i.e. arresting the development of a disease state orcondition, and ameliorating, i.e. causing regression or delayingprogression of a disease. Treatment can be to ameliorate diseasesymptoms without curing a patient.

The term “preventing” or “prevention” as used herein does not mean tostop a disease state or condition from occurring in a patient or subjectcompletely but may also refer to a reduced risk of developing a diseasestate or condition.

The present invention is further illustrated by the following examples,without being necessarily limited to these embodiments of the invention.An example or part thereof, including compounds, doses andadministration routes, as well as treatment combinations, each as suchor in combination with the detailed description above forms part of theinvention.

EXAMPLES Acronyms and Abbreviations

-   CD Cluster of differentiation-   DMEM Dulbecco's Modified Eagle Medium-   FCS Fetal calf serum-   ID Identifier-   i.p. intraperitoneal-   LAG-3 Lymphocyte activation gene 3-   M Molar-   MEM Minimum Essential Media-   PBS Phosphate buffered saline-   PD-1 Programmed cell death 1-   PD-L1 Programmed cell death ligand 1-   PD-L2 Programmed cell death ligand 2-   q3or4d every 3^(rd) or 4^(th) day (twice weekly)-   R Response-   TGI Tumour Growth Inhibition, calculated to the formula:

TGI=100×{1−[(treatedfinal day−treatedday1)/(controlfinalday−controlday1)]}

-   TIL Tumour infiltrating lymphocytes

Example 1 Test Compounds

A rat IgG2a anti-murine PD-1 antibody EX101359 (clone RMP1-14) (10mg/kg), the bispecific, VEGF and Ang2 binding protein VEGFANGBII22(having a structure/sequence as defined hereinabove and in SEQ IDNOs:11, 12 and 13), the small molecule TKI vatalanib and the CrossMabantibody vanucizumab (anti-human VEGF-A and anti-human/murine Ang-2)were used for these experiments. For the control group the correspondingIsotype to the PD-1 antibody Rat IgG2a (EX101362) was used (10 mg/kg),i.e. an antibody which has a similar sequence but does not bind to PD-1.All antibodies were diluted in 1×PBS and were ordered from BioXcell,West Lebanon, N.H., USA.

Cells

LL/2 (LLC1) is a murine lung carcinoma cell line, which was bought fromAmerican Type Culture Collection (ATCC), USA (CRL-1642). Cells werecultured in T175 tissue culture flasks at 37° C. and 5% CO₂. The mediumused was DMEM supplemented with 10% FCS (HyClone® Fetal Bovine SerumCharacterized; Cat No SH30071.03; by Thermo Scientific). Cultures weresplit every two-three days with a ratio of 1:4/1:6.

Mice

The C57BL/6NTac is a fully immune competent mouse strain, which wassupplied by Taconic Denmark. Female mice were shipped from Taconic at anage of 7 weeks to a local animal facility. After arrival at the animalfacility, mice were allowed to adjust to conditions at least for 5 daysbefore they were used for experiments. They were housed in groups of 10under standardized conditions at 21.5+/−1.5° C. temperature and 55+/−10%humidity. Standardized diet (PROVIMI KLIBA) and autoclaved tap waterwere provided ad libitum. Subcutaneous microchips implanted underisoflurane anesthesia were used to identify each mouse. Cage cardsshowing the study number, the animal identification number, the antibodyand compound and dose levels, the administration route as well as theschedule remained with the animals throughout the study.

Establishment of Tumors, Randomization

The animals were dispatched randomly by the computer program SEPIA intothe different groups. To establish the subcutaneous tumor model, LL/2cells were harvested, resuspended in PBS/5% FCS and mixed 1:2 withMatrigel® Matrix (without growth factors, Corning, Tewksbury, Mass.,USA) at 5×10⁵/ml. 100 μl cell suspension containing 5×10⁴ cells wereinjected per mouse (day-5).

Administration of Test Compound

Antibodies were diluted in PBS to 1 mg/mL and injected intraperitoneallywith a volume of 10 ml/kg. Dilution was kept at 4° C. for a maximum of 5days. Start of treatment (day 1) was 3 days post cell injection.Vatalanib was suspended in a 5% Natrosol™ hydroxyethylcellulose solutionand administered intragastrally by gavage needle (Infusionskanüle OliveA, Acufirm, No. 14 64 ll-1). The administration volume was 10 ml per kgbody weight. Administration was once every 24 h. To prepare thesuspension, compound was added to the Natrosol™ hydroxyethylcellulosesolution and the mixture was stirred overnight. Sometimesultrasonication was needed. Solutions were kept at room temperature inthe dark for a maximum of 1 week.

TABLE 2 Treatment groups Group No. of mice Treatment compound(s) Dose[mg/kg] Schedule Route 1 10 Isotype PD-1 antibody (EX101362) 10 mg/kgq3or4d i.p. 2 10 anti-murine PD-1 antibody (EX101359) 10 mg/kg q3or4di.p. 3 10 VEGFANGBII22 15 mg/kg q3or4d i.p. 4 10 Vatalanib (EXBF003) 100mg/kg  qd po 5 10 anti-murine PD-1 antibody (EX101359); 10 mg/kg,q3or4d, i.p., VEGFANGBII22; 15 mg/kg, qd p.o. Vatalanib (EXBF003) 100mg/kg  6 10 anti-VEGF/Ang2 antibody (vanucizumab); 15 mg/kg q3or4d i.p.7 10 anti-murine PD-1 antibody (EX101359); 10 mg/kg, q3or4d, i.p.,anti-VEGF/Ang2 antibody (vanucizumab); 15 mg/kg, qd p.o. Vatalanib(EXBF003) 100 mg/kg 

Monitoring Tumor Growth and Disease Progression

Tumor diameters were measured three times a week (Monday, Wednesday andFriday) with a caliper. From day 14 until day 28 after the start oftreatment, all tumors were measured daily. The volume of each tumor [inmm³] was calculated according to the formula “tumorvolume=length*diameter²*pi/6.” To monitor side effects of treatment,mice were inspected daily for abnormalities and body weight wasdetermined at least three times a week. Animals were sacrificed when thetumors reached a size of 1500 mm³ or a tumor necrosis was bigger than ⅓of the tumour surface. In addition, animals with a body weight loss >18%were euthanized for ethical reasons. Tumor growth inhibition (TGI)values were calculated as follows:

TGI=100×{1−[(treated_(final day)−treated_(day1))/(control_(final day)−control_(day1))]}

Statistical Analysis—Anti-Tumor Efficacy

For the evaluation of the statistical significance of tumor inhibition aone-tailed nonparametric Mann-Whitney-Wilcoxon U-test was performed,based on the hypothesis that an effect would only be measurable in onedirection (i.e. expectation of tumor inhibition but not tumorstimulation). In this case, the U-test compares the ranking of theindividual tumors of two groups, according to the absolute volume on aparticular day (pairwise comparisons between groups). Analysis wasperformed on day 17 of the experiment. The p-values obtained from theU-test were adjusted using the Bonferroni-Holm correction. Byconvention, p-values ≤0.05 indicate significance of differences.

Results

Mice in the isotope control group were treated twice weekly withEX101362 intraperitoneally. During the treatment period tumors grew froma median volume of 41 mm³ to a volume of 1275 mm³. Treatment with 10mg/kg EX101359, the anti-murine PD-1 antibody, was administered twiceweekly intraperitoneally. EX101359-treatment reduced tumour growthcompared to the isotype control (median TGI=51%).

Treatment with VEGFANGBII22 alone resulted in a TGI of 49%. Treatmentwith vatalanib alone resulted in a TGI of 54%. The combined treatmentwith vatalanib, VEGFANGBII22 and anti-PD1 resulted in a TGI of 79%.

Treatment with vanucizumab alone resulted in a TGI of 64%. The combinedtreatment with vatalanib, vanucizumab and anti-PD1 resulted in a TGI of81%.

Further results of the combination and results of comparative compoundsare shown in FIG. 1.

CONCLUSION

In this study the monotherapies of anti-PD1, vatalanib (targetingVEGFR), VEGFANGBII22 and vanucizumab showed a minimal to mediumanti-tumor response with TGI values of 51%, 54%, 49% and 64%,respectively. The triple drug combinations of anti-PD1 plus vatalanibplus VEGFANGBII22 or anti-PD1 plus vatalanib plus vanucizumab wereclearly more effective with TGI values of 79% and 81%. By analyzing theresponse curves of the individual tumors the triple drug combination ofanti-PD1 plus vatalanib plus VEGFANGBII22 stand out as one of thetreated tumors shows shrinkage whereas all tumors in the anti-PD1 plusvatalanib plus vanucizumab treated group show reduced tumor growth.

The triple drug combinations show a better anti-tumor effect compared tothe isotype control and the monotherapies. The triple drug combinationof anti-PD1 plus vatalanib plus the VEGFANGBII22 even induced tumorshrinkage in one treated individual. All treatments were well toleratedin this experiment.

It should be understood from the above that upon transferring theseresults to a therapy in humans, the compound VEGFANGBII22 will includean anti-Ang2 as well as an anti-VEGF activity, as this compound isbinding to human VEGF. Thus, additional use of an anti-VEGF agent suchas vatalanib in the above-described experiment will not be required.Thus, the above data indicate that treatment of humans with VEGFANGBII22plus a PD1 antagonist, such as an anti-PD1 antibody, will bring aboutuseful therapeutic effects in human patients.

Example 2

A further experiment was set up essentially as described in Example 1,comparing monotherapies with the triple drug combination VEGFANGBII22plus anti-PD1 antibody plus vatalanib (with vatalanib again mimickingthe anti-VEGF activity in mice, which activity cannot be expected fromVEGFANGBII22 because the VEGF binding component of that compound doesnot bind to mouse VEGF).

Again, TGI values were determined, with 80% being achieved uponadministration of the triple combination. In contrast thereto, TGIvalues of 22% (anti-PD-1 antibody), 27% (anti-Ang2 activity ofVEGFANGBII22) and 43% (vatalanib) have been achieved by themonotherapies.

Additionally, survival of the mice within the respective treatment grouphas been determined. As can be seen from FIG. 2, only treatment withVEGFANGBII22, vatalanib and the PD1 antagonist provided for survivalssignificantly beyond the 30 days limit. Again, this shows that theproposed treatment may be expected to achieve superior effects also inhumans (again being understood that the anti-VEGF activity will alreadybe provided by the VEGFANGBII22 component, so that no addition ofvatalanib will be required).

1. A pharmaceutical composition for the treatment of solid tumor cancercomprising a) a Compound A and b) a Compound B wherein Compound A is abinding molecule comprising a VEGF-binding immunoglobulin singlevariable domain, a serum albumin binding immunoglobulin single variabledomain, and an Ang2-binding immunoglobulin single variable domainwherein said VEGF-binding immunoglobulin single variable domain has thefollowing CDR sequences: CDR1: (SEQ ID NO: 1) SYSMG, CDR2:(SEQ ID NO: 2) AISKGGYKYDAVSLEG, CDR3: (SEQ ID NO: 3) SRAYGSSRLRLADTYEY;

said serum albumin binding immunoglobulin single variable domain has thefollowing CDR sequences: CDR1: (SEQ ID NO: 4) SFGMS, CDR2:(SEQ ID NO: 5) SISGSGSDTLYADSVKG, CDR3: (SEQ ID NO: 6) GGSLSR;

said Ang2-binding immunoglobulin single variable domain has thefollowing CDR sequences: CDR1: (SEQ ID NO: 7) DYAIG, CDR2:(SEQ ID NO: 8) AIRSSGGSTYYADSVKG, CDR3: (SEQ ID NO: 9)VPAGRLRYGEQWYPIYEYDA;

and wherein each single variable domain is separated by a linkerpeptide; and wherein Compound B is a PD-1 antagonist selected from thegroup consisting of pembrolizumab, nivolumab, pidilizumab, PD1-1 (HC/LC;SEQ ID NO: 14, 15), PD1-2 (HC/LC; SEQ ID NO: 16, 17), PD1-3 (HC/LC; SEQID NO: 18, 19), PD1-4 (HC/LC; SEQ ID NO: 20, 21), and PD1-5 (HC/LC; SEQID NO: 22, 23).
 2. The pharmaceutical composition according to claim 1,wherein said immunoglobulin single variable domains of Compound A areVHH domains.
 3. The pharmaceutical composition according to claim 1,wherein Compound A has the amino acid sequence according to SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO:
 13. 4. The pharmaceutical compositionaccording to claim 1, further comprising one or more pharmaceuticallyacceptable carriers, excipients and/or vehicles.
 5. The pharmaceuticalcomposition according to claim 1, wherein the solid tumor cancer is lungcancer.
 6. The pharmaceutical composition according to claim 5, whereinthe solid tumor cancer is non-small cell lung cancer (NSCLC).
 7. A kitcomprising a) a first pharmaceutical composition comprising Compound Aand b) a second pharmaceutical composition comprising Compound B,wherein Compound A is a binding molecule comprising a VEGF-bindingimmunoglobulin single variable domain, a serum albumin bindingimmunoglobulin single variable domain, and an Ang2-bindingimmunoglobulin single variable domain wherein said VEGF-bindingimmunoglobulin single variable domain has the following CDR sequences:CDR1: (SEQ ID NO: 1) SYSMG, CDR2: (SEQ ID NO: 2) AISKGGYKYDAVSLEG, CDR3:(SEQ ID NO: 3) SRAYGSSRLRLADTYEY;

said serum albumin binding immunoglobulin single variable domain has thefollowing CDR sequences: CDR1: (SEQ ID NO: 4) SFGMS, CDR2:(SEQ ID NO: 5) SISGSGSDTLYADSVKG, CDR3: (SEQ ID NO: 6) GGSLSR;

said Ang2-binding immunoglobulin single variable domain has thefollowing CDR sequences: CDR1: (SEQ ID NO: 7) DYAIG, CDR2:(SEQ ID NO: 8) AIRSSGGSTYYADSVKG, CDR3: (SEQ ID NO: 9)VPAGRLRYGEQWYPIYEYDA;

and wherein each single variable domain is separated by a linkerpeptide; and wherein Compound B is a PD-1 antagonist selected from thegroup consisting of pembrolizumab, nivolumab, pidilizumab, PD1-1 (HC/LC;SEQ ID NO: 14, 15), PD1-2 (HC/LC; SEQ ID NO: 16, 17), PD1-3 (HC/LC; SEQID NO: 18, 19), PD1-4 (HC/LC; SEQ ID NO: 20, 21), and PD1-5 (HC/LC; SEQID NO: 22, 23).
 8. The kit according to claim 7, wherein saidimmunoglobulin single variable domains of Compound A are VHH domains. 9.The kit according to claim 7, wherein Compound A has the amino acidsequence according to SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.10. The kit according to claim 7, further comprising a package insertcomprising readable instructions for simultaneous, concurrent,sequential, successive, alternate or separate administration to apatient in the treatment of solid tumor cancer.
 11. The kit according toclaim 10, wherein the solid tumor cancer is lung cancer.
 12. The kitaccording to claim 11, wherein the solid tumor cancer is non-small celllung cancer (NSCLC).